The Problem: Mesothelioma is a devastating cancer where the majority of patients will die from their disease. The last significant advance in chemotherapy for mesothelioma was pemetrexed, which was approved by the FDA in 2004. The vast majority of mesothelioma tumors have defects in the cell cycle. Low expression of the CDK4/CDK6 inhibitor p16INK4a has been demonstrated in multiple basic and clinical studies of mesothelioma. A phase III study reported a significant increase in survival in breast cancer patients who received palbociclib, a selective CDK4/6 inhibitor. Mesothelioma tumors also exhibit activation of the PI3K/MTOR pathway, and inhibition of PI3K/MTOR has been demonstrated to overcome CDK4/6 inhibitor resistance in breast cancer cells. A decrease in reactive oxygen species (ROS) by mitochondrial antioxidant defense proteins plays a key role in the ability of mesothelioma cells to overcome the pro-apoptotic signaling induced by ROS. Knowledge to be Addressed: The long-term goal of this laboratory is to identify key principles of cancer biology that will allow the appropriate selection of the most effective combination therapies based on upfront molecular signatures. The objective of this application as a next step in the pursuit of this long-term goal is to determine how targeting CDK4/6 may be exploited in combination with inhibition of CDK2, PI3K/MTOR, or mitochondrial antioxidant defense in the treatment of mesothelioma. Findings will be correlated with mRNA expression signatures that can be applied for to selecting treatment combinations for patients. Hypothesis: Our central hypothesis is that inhibition of CDK4/6 in combination with inhibition of CDK2, PI3K/MTOR, or mitochondrial antioxidant defense will cause cell death for multiple and distinct molecular subtypes of mesothelioma. Preliminary Studies: We have demonstrated that mesothelioma cell proliferation can be decreased via Cyclin D1 knockdown and small molecule inhibition of CDK4/6. We have also demonstrated that gentian violet (known to inhibit antioxidant defense) sensitizes mesothelioma cells to palbociclib in a synergistic fashion. Specific aims: 1) Identify mechanisms by which CDK4/6 contributes to mesothelioma cell survival, determine whether resistance to CDK4/6 inhibition varies by molecular subtype, and determine whether simultaneous inhibition of CDK2 has synergistic benefits compared to inhibition of CDK4/6 alone. 2) Define the mechanism by which PI3K/MTOR overcomes cell cycle inhibition and identify how the effectiveness of simultaneous inhibition of CDK4/6 and PI3K/MTOR in mesothelioma varies by molecular subtype. 3) Identify mechanisms by which inhibition of mitochondrial antioxidant defense and CDK4/6 individually or in combination affect mitochondrial function and regulated cell death in mesothelioma cells. Significance and Innovation: The proposed work is innovative because it capitalizes on evaluation of agents for their ability to affect the cell cycle, PI3K/MTOR, or antioxidant defense in combination in mesothelioma based on molecular profiling in a cancer known to be refractory to therapy. Based on the genomic profile of mesothelioma, using cell cycle inhibition as a component of combination therapy should be ideal. The proposed research will lay a groundwork for how cell cycle inhibitors can be given in combination with inhibitors of CDK2, PI3K/MTOR, or mitochondrial antioxidant defense. A very positive translational impact of this work is that it will inform the design of clinical trials for testing CDK4/6 inhibitors in combination with other active cancer-fighting drugs.